The epidermis performs essential barrier functions that protect the body from environmental insults and maintain fluid-electrolyte balance. Defects in epidermal and follicular development lead to numerous diseases such as skin cancer, psoriasis, atopic dermatitis, alopecia, and impaired wound healing. Whereas transcriptional programs have been well-studied in development and diseases of the skin, the role of alternative splicing (AS) in skin development and function is essentially unstudied. Nearly all mammalian multi- exon genes produce AS mRNAs and tissue-specific AS factors coordinate programs of AS to regulate biologically coherent pathways. Moreover, our preliminary data reveals that AS plays a critical, but unappreciated role in the regulatory programs of the development and function of both skin and hair. In this proposal, we will use our unique genetic mouse models to study the critical roles of splicing factors identified in our lab in epidermal physiology and hair growth. My laboratory made a landmark discovery that epithelial cell- type-specific splicing factors Esrp1 and Esrp2 regulate fibroblast growth factor receptor 2 (Fgfr2) splicing, an AS event previously implicated in skin development and epidermal barrier function. To further investigate the role of Esrp-dependent AS in skin development, we generated mice with conditional and complete knockout alleles for Esrp1 and Esrp2. Combined Esrp1/Esrp2 KO is lethal and results in epidermal hypoplasia, defects in epidermal barrier function, and reduced numbers of hair follicles. We hypothesize that these phenotypes reflect the loss of key epithelial-specific splice isoforms and will use conditional gene knockout technology to further characterize the phenotypes of Esrp deletion and define a genome-wide program of Esrp-regulated AS in the skin through the following aims: 1) Determine the phenotypes associated with Esrp ablation in the interfollicular epidermis and in hair follicles. We will conditionally abate the Esrps using Esrp1flox/flox/Esrp2-/- mice in developing and adult epidermis and characterize the basic cellular processes that lead to epidermal and follicular defects. 2) Define comprehensive programs of Esrp regulated alternative splicing in the epidermis. We will use RNA-Seq and splicing sensitive microarrays to define genome-wide programs of AS in vitro and in vivo. Esrp regulated targets in the epidermis will be functionally screened in epithelial barrie assays. 3) Identify Esrp-regulated splicing programs in the hair follicle and differential splicingin the dermal papilla (DP). We will use inducible deletion strategies to determine the consequence of Esrp ablation on hair follicles and identify key Esrp targets in hair follicle bulge stem cells nd the hair germ (HG). The proposed aims constitute the first comprehensive analysis of AS in skin development and function, thereby introducing a new paradigm to the field. These studies are needed to define the molecular and cellular mechanisms by which Esrp ablation in the skin induces epidermal barrier defects and hair loss in order to inform the development of future therapies to treat skin pathologies and alopecia.